Aqueous humor immune factors and cytomegalovirus (CMV) levels in CMV retinitis through treatment – The CRIGSS study

Cytokine 84 (2016) 56–62

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Aqueous humor immune factors and cytomegalovirus (CMV) levels in CMV retinitis through treatment – The CRIGSS study Jayant Venkatramani Iyer a,b,⇑,1, Rupesh Agrawal a,1, Tun Kuan Yeo a, Dinesh V. Gunasekeran a, Praveen Kumar Balne a, Bernett Lee c, Veonice Bijin Au c, John Connolly c,d, Stephen C.B. Teoh a a

National Healthcare Group Eye Institute, Tan Tock Seng Hospital, Singapore Singapore National Eye Center, Singapore Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore d Institute of Biomedical Studies, Baylor University, Waco, TX, USA b c

a r t i c l e

i n f o

Article history: Received 23 December 2015 Received in revised form 29 March 2016 Accepted 10 May 2016 Available online 27 May 2016 Keywords: Aqueous humor Cytomegalovirus HIV Immune privilege Opportunistic infection

a b s t r a c t Purpose: This study aims to perform comprehensive longitudinal immune factor analysis of aqueous humor in relation to the aqueous CMV viral load and systemic CD4 counts during treatment of patients with co-infection of HIV and CMVR. Methods: Aqueous humor samples were collected from 17 HIV-positive patients with CMVR scheduled to undergo weekly intravitreal ganciclovir therapy as part of the prospective CMV Retinitis Intravitreal Ganciclovir Singapore Study (CRIGSS) over the course of 1 year. Full data across all the 4 time points was obtained and analyzed for CMV DNA viral load, 41 cytokine and chemokine factors using realtime PCR with the FlexMAP 3D (LuminexÒ) platform and assessed using the Milliplex Human CytokineÒ kit. Results: The following immune factors (Spearman correlation coefficient r value in parenthesis, p < 0.05) showed strong correlation with CMV DNA load in the aqueous - MCP-1 (0.80, IFN-g (0.83), IP-10 (0.82), IL-8 (0.81), fractalkine (0.73), RANTES (0.68) - while the following showed moderate correlation - PDGFAA (0.58), Flt-3L (0.59) and G-CSF (0.53). Only PDGF-AA revealed a statistically significant negative correlation with serum CD4 levels (r = 0.74). Conclusion: Immune factors that correlate with intraocular CMV DNA load are identified. They are indicative of a Th1 and monocyte-macrophage mediated response, and exhibit a decreasing trend longitudinally through the course of treatment. These factors may be an important new consideration in individualizing the treatment of patients with CMVR. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction The Human Immunodeficiency Virus (HIV) establishes its infection in a host by hijacking host immunity through immune and metabolic dysregulation [1]. Acquired Immune Deficiency Syndrome (AIDS) is the late stage of the prolific HIV infection that heralds the onset of a myriad of possible opportunistic infections due to the severely depressed cellular immunity of the host in AIDS [1]. Cytomegalovirus retinitis (CMVR) is the most common opportunistic ocular infection in patients with acquired immune deficiency syndrome (AIDS) when CD4 counts are <50 cells/lL [2–4]. ⇑ Corresponding author at: Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751, Singapore. E-mail address: [email protected] (J.V. Iyer). 1 Joint first authors. http://dx.doi.org/10.1016/j.cyto.2016.05.009 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.

Before the introduction of highly active antiretroviral therapy (HAART) in 1996, 25–42% of AIDS patients developed CMVR [2,3,5]. While there has been an 80% decrease in the incidence of CMVR since the introduction of HAART, CMVR still remains the most common cause of vision loss in patients with AIDS [2,6]. CMV infections are also a major concern in other patients with depressed cellular immunity from other causes, including those with hematological malignancies or immunosuppressive treatment post-organ transplantation [7]. However, these patients have a reportedly greater risk of life-threatening CMV pneumonitis [8] while HIV patients are more susceptible to CMVR [9]. In patients with HIV, treatment of CMVR with anti-CMV medications is often based on the CD4 counts as a surrogate measure of the patient’s immune status when on HAART [10]. In non-HIV patients, monitoring is more complicated and subjective. Assessment is clinical, based on the overall ‘impression’ of the patient’s systemic

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condition in consult with the internist and quiescence of CMV inflammation in the eye as determined by the ophthalmologist [11]. The immunopathology of uveitis and ocular inflammation is mediated by numerous inflammation-related mediators wherein over- or under-expression of various cytokines determines the outcome of disease processes [12]. These intraocular mediators can be found within intraocular fluids such as the aqueous and vitreous humor, and reflect the state of inflammation within the eye [13]. There have been a few reports that associate different cytokine profiles with different forms of uveitides. Cytokine profile patterns may serve as diagnostic and prognostic monitoring tools for the clinician [12–14]. In our recently published study, we comprehensively analyzed the cytokine, chemokine and growth factor levels in the aqueous humor of CMVR patients and found a unique immunological signature consisting of raised levels of IP-10, fractalkine, PDGF-AA, G-CSF and Flt-3L [15]. In this study we aimed to perform prospective longitudinal analysis of aqueous immune factor levels through the course of treatment with intravitreal ganciclovir in AIDS patients with CMVR who were placed on concomitant highly active anti-retroviral therapy (HAART). Aqueous CMV viral load was also assessed in relation to aqueous immune factor levels and serum CD4 counts throughout treatment. This study may lead to identification of novel indicators for targeted therapeutic intervention in the management of CMVR, and for monitoring of disease progression and prognostication. 2. Methodology We recruited 17 consecutive HIV-positive patients with their informed consent over a one-year period. All HIV-positive patients were reviewed by the Uveitis Service at the Communicable Disease Center (CDC), Tan Tock Seng Hospital. Approval was obtained from the Tan Tock Seng Hospital Institutional Review Board (IRB) (A/10/465). Subjects who were HIV-positive were confirmed by

Table 1 Demographic details of HIV patients clinically diagnosed with CMV retinitis. Patients demographic details (n = 17) Ethnicity Chinese Malay Indian Others

13/17 (76.5%) 2/17 (11.8%) 1/17 (5.9%) 1/17 (5.9%)

Mean age

49.5 years (range: 35–63 years)

Male female ratio

16:1

Western blot (Diagnostic Biotechnology HIV Blot 2.2), with CMV retinitis diagnosed clinically – characterized by necrotizing retinitis with edematous or granular white borders, with or without associated hemorrhage [3], typically with minimal or no clinical vitritis or anterior chamber inflammation. Though the diagnosis of CMVR is traditionally clinical, and investigations are usually unnecessary [16] aqueous humor samples from eight HIV patients were tested for CMV DNA by qPCR and CD4 count assay was done on patients’ blood samples using Trucount tubes (BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions. All patients underwent HAART as well as weekly intravitreal ganciclovir injections (irrespective of their CMV PCR results) as part of the CRIGSS study. 2.1. Aqueous humor sampling Anterior chamber paracentesis for patients with CMVR undergoing treatment with intravitreal ganciclovir was performed under topical anesthesia and direct visualization with patients in an inclined position, using an aseptic technique. Aqueous samples were drawn using a 30-gauge needle attached to an insulin syringe, which was inserted into the anterior chamber through the clear cornea in a plane above and parallel to the iris. Patients were prescribed topical antibiotics for a week following the procedure. At least 100 lL of aqueous was drawn from all patients. The

2500 2250 2000 1750 1500 1250 1000

Copies/ul

750 500 250 50 40 30 20 10

003 CC 004 CC 005 CC 006 CC 001 KHW 002 KHW 003 KHW 004 KHW 005 KHW 006KHW 001 THP 002 THP 003 THP 004 THP 005 THP 001 TS 001 LMK 002 LMK

001 TCL 002 TCL 003 TCL 004 TCL 005 TCL 006 TCL 007 TCL 008 TCL 009 TCL 010 TCL 011 TCL 012 TCL 001 HBM 002 HBM 004 HBM 005 HBM 001 LLH 002 LLH 004 LLH 005 LLH 001 CC 002 CC

0

Sample ID Fig. 1. CMV viral load in aqueous humor samples collected from HIV patients at different time intervals.

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2.3. Statistical analysis

samples were then stored in ice and sent to the A⁄Star immunology laboratory for analysis within 4 h of isolation to preserve protein integrity. Samples were fractionated prior to further comprehensive cytokine and growth factor analysis.

Data preparation was done in Accelrys Pipeline Pilot and statistical analysis done in the R statistical language (version 2.12.1). Data visualization was done in TIBCO Spotfire. Statistical significance was set at a p-value of less than 0.05 with Bonferroni correction performed for multiple testing. As for the statistical design, based on our results and testing procedure, the hypothesis is that there are significant associations of Luminex measurements with progression or other clinical measurements. The statistical test would then test for the null hypothesis which is that there are no significant associations between Luminex measurements and progression or clinical measurement. The alternative hypothesis is that there is a significant association. Correlation of the immune factor levels (pg/mL) with time (weeks) was performed using the Spearman Rank correlation. Associations between Luminex concentrations and time (weeks) were analyzed using Spearman Rank correlation. All available data were included in this analysis (including patients who did not

2.2. Aqueous analysis Aqueous humor fractions were analyzed for the concentration of 41 different cytokines, chemokines and growth factors perceived to be involved in viral induced inflammation, with the FlexMAP 3D (LuminexÒ) platform using the Milliplex Human CytokineÒ kit. Aqueous humor samples were drawn from these subjects pretreatment and at 4-weekly intervals through the 12-week (or more) course of intravitreal ganciclovir treatment. Aqueous humor was stored in ice and sent to the lab within 4 h for analysis. 41 cytokine and chemokine factors were measured in the lab using FlexMAP 3D (LuminexÒ) platform and assessed using the Milliplex Human CytokineÒ kit [17]. Eight subjects also had aqueous CMV DNA viral load assessed [17].

Fractalkine

G−CSF

IFN−g

100

100

10.0

10

1.0

10 1 0.1

Analyte concentration (pg/ml)

IL−10

IL−12p70

IL−6

10

1000

10.0

10 0.1

1

IL−8

IP−10

MCP−1

10000 10000

100

100 1000

1

0

2

4

6

8

10

12

0

2

4

6

8

10

12

0

2

4

Weeks Fig. 2. Distribution plot of analytes concentration in aqueous humor (pg/mL) over weeks at four time points.

6

8

10

12

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complete treatment) due to the small sample size. The significance of the Spearman Rank correlation was computed from the tdistribution with p-values <0.05 deemed to be significant. 3. Results Of the seventeen subjects, only 9 completed 12 weeks of treatment, while 10 completed up to 8 weeks of treatment under the study. Demographic details of HIV patients clinically diagnosed with CMV retinitis were mentioned in Table 1. Out of 17 patients, eight patients aqueous humor samples were tested for the presence of CMV DNA using qPCR. Of the eight, seven patients were found positive for CMV DNA and among them six patients’ aqueous humor was serially analyzed for CMV viral load up to 12 weeks of ganciclovir treatment (Fig. 1).

defaulted on his appointments. He had persistently depressed CD4 counts (<100/mm3) but clinically quiescent CMVR. Subsequent analysis of immune factor trending was performed after omitting this outlier. 9 of the immune factors showed a statistically significant negative correlation for samples analyzed longitudinally over the 4 times points – G-CSF, IL-8, IFN-g, IP-10, fractalkine, IL-12p70, MCP-1, IL-10 and IL-6 (Fig. 2 and Table 2). 9 of the immune factors (Spearman coefficient, r, in brackets) – MCP-1 (0.80), IFN-g (0.83), IP-10 (0.82), IL-8 (0.81), fractalkine (0.73), RANTES (0.68), PDGF-AA (0.58), FLT-3L (0.59) and G-CSF (0.53) – showed statistically significant positive correlation with CMV DNA viral load through course of treatment (Table 3 and Fig. 3). Only aqueous PDGF-AA levels (r = 0.74) showed a statistically significant negative correlation with serum CD4 levels through treatment.

3.1. Aqueous humor cytokine analysis 4. Discussion Of the 41 analytes investigated in the aqueous humor samples, 27 were expressed at measurable levels of >0.8 pg/mL. Measurements below the detection limits were assigned a value of 0.1 for the purpose of statistical computations. One of the 17 subjects was an outlier for all the tested immune factor levels as there was no significant difference in the levels throughout the course of treatment across the pre-treatment and 3 treatment time points. Clinical review of this patient revealed resistance to HAART therapy, and that the patient frequently

Our previous study revealed a unique immunological signature in aqueous humor of eyes with CMVR suggestive of a combined Th1-lymphocyte and monocyte-macrophage driven immune response with a unique immunological signature [15]. This study tracks immune factor levels through the course of treatment in HIV-positive CMVR patients, and aims to identify markers suggestive of good treatment response. The decrease in abovementioned intraocular immune factor levels coincident with

Table 2 Aqueous immune factor levels (pg/mL) correlated with number of weeks on treatment, using Spearman Rank correlation2.

⁄

Analyte

N

SR Rho

G-CSF IL-8 IFN-g IL-6 MCP-1 IL-10 IP-10 IL-15 TNF-b IL-1a PDGF-BB IL-17 GRO MIP-1a RANTES IL-3 MCP-3 IL-2 IL-9 IL-12p40 PDGF-AA IL-4 Flt-3L VEGF Fractalkine EGF FGF-2 IL-12p70 sCD40L MIP-1b IL-13 IFN-a2 IL-5 IL-1ra IL-7 TNF-a IL-1b Eotaxin TGF-a MDC

42 42 34 42 34 34 42 34 34 34 34 34 42 34 34 34 34 34 34 34 42 34 42 34 42 34 34 34 34 42 34 34 34 34 34 34 34 34 34 34

0.61 0.53 0.50 0.47 0.47 0.47 0.47 0.37 0.36 0.34 0.33 0.33 0.32 0.31 0.30 0.29 0.23 0.23 0.21 0.19 0.17 0.17 0.15 0.15 0.14 0.13 0.12 0.10 0.10 0.10 0.08 0.08 0.06 0.06 0.06 0.03 0.03 0.03 0.01 0.01

Statistically significant.

SR Rho squared

p value

p value (corrected)

0.37 0.28 0.25 0.22 0.22 0.22 0.22 0.14 0.13 0.12 0.11 0.11 0.10 0.09 0.09 0.09 0.05 0.05 0.04 0.04 0.03 0.03 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

2.14E 2.96E 2.67E 1.56E 5.10E 5.14E 1.82E 2.96E 3.87E 4.94E 5.38E 5.59E 3.90E 7.94E 8.34E 9.26E 1.84E 1.86E 2.44E 2.82E 2.85E 3.43E 3.37E 4.01E 3.71E 4.78E 5.08E 5.73E 5.75E 5.49E 6.41E 6.59E 7.50E 7.53E 7.55E 8.54E 8.66E 8.86E 9.43E 9.69E

8.56E 5.91E 2.13E 1.82E 2.94E 2.94E 1.82E 1.48E 1.56E 1.72E 1.72E 1.72E 1.56E 2.22E 2.22E 2.31E 4.12E 4.12E 5.14E 5.44E 5.44E 5.96E 5.96E 6.41E 6.18E 7.36E 7.53E 7.67E 7.67E 7.67E 8.24E 8.24E 8.62E 8.62E 8.62E 9.32E 9.32E 9.32E 9.67E 9.69E

05 04 03 03 03 03 03 02 02 02 02 02 02 02 02 02 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

04⁄ 03⁄ 02⁄ 02⁄ 02⁄ 02⁄ 02⁄ 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

Mean (pg/mL)

SEM

38.21 16.41 3.72 51.56 3099.27 5.23 892.27 1.42 1.33 1.26 12.69 1.18 29.50 7.56 8.06 1.14 26.48 1.12 1.47 3.04 5.38 1.21 15.50 29.38 67.95 2.52 9.75 1.65 9.99 5.79 0.39 3.63 3.05 4.04 1.81 0.62 0.35 2.15 1.59 13.07

12.00 10.84 1.29 28.72 697.02 1.08 337.72 0.34 0.63 0.20 3.15 0.07 14.90 1.18 1.71 0.27 6.03 0.93 0.53 0.99 1.28 0.36 2.28 7.30 13.39 0.41 2.30 0.44 3.66 1.03 0.09 0.90 2.00 1.72 0.31 0.11 0.11 0.54 0.01 1.99

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Table 3 Aqueous immune factor levels (pg/mL) correlated with viral load, using Spearman Rank correlation2. Analyte IFN-g IP-10 PDGF-BB IL-8 MCP-1 IL-4 Fractalkine RANTES IL-15 MIP-1b EGF IL-9 IL-10 MIP-1a Flt-3L PDGF-AA IFN-a2 MCP-3 G-CSF IL-6 IL-2 TNF-a sCD40L GRO IL-1b IL-5 IL-1ra IL-13 MDC TNF-b VEGF ⁄

N 29 32 8 29 38 4 20 38 7 19 4 5 24 19 33 37 18 21 36 24 17 35 11 20 6 38 11 26 25 14 19

SR Rho 0.83 0.82 0.81 0.81 0.80 0.74 0.73 0.68 0.67 0.65 0.60 0.60 0.60 0.60 0.59 0.58 0.56 0.56 0.53 0.53 0.52 0.46 0.38 0.36 0.36 0.35 0.31 0.30 0.25 0.23 0.13

SR Rho squared 0.69 0.68 0.66 0.66 0.64 0.54 0.54 0.47 0.44 0.42 0.36 0.36 0.36 0.36 0.35 0.34 0.31 0.31 0.28 0.28 0.27 0.21 0.14 0.13 0.13 0.12 0.09 0.09 0.06 0.05 0.02

p value 2.60E 6.85E 1.48E 1.06E 2.06E 2.62E 2.43E 2.14E 1.02E 2.55E 4.17E 3.50E 2.03E 6.87E 2.86E 1.50E 1.56E 8.27E 8.02E 9.15E 3.18E 5.58E 2.48E 1.18E 4.83E 3.27E 3.62E 1.35E 2.33E 4.32E 5.88E

08 09 02 07 09 01 04 06 01 03 01 01 03 03 04 04 02 03 04 03 02 03 01 01 01 02 01 01 01 01 01

p value (corrected) 2.69E 1.06E 2.84E 8.23E 6.40E 3.25E 1.08E 1.33E 1.58E 7.19E 4.61E 4.15E 6.29E 1.64E 1.11E 7.73E 2.84E 1.83E 2.76E 1.89E 5.34E 1.44E 3.20E 1.75E 4.99E 5.34E 4.15E 1.91E 3.14E 4.62E 5.88E

⁄

07 07⁄ 02⁄ 07⁄ 08⁄ 01 03⁄ 05⁄ 01 03⁄ 01 01 03⁄ 02⁄ 03⁄ 04⁄ 02⁄ 02⁄ 03⁄ 02⁄ 02 02⁄ 01 01 01 02 01 01 01 01 01

Mean (pg/mL)

SEM

4.50 1462.78 13.18 22.52 3300.38 0.63 93.85 6.81 1.46 8.43 2.15 0.27 5.80 6.31 11.40 7.73 3.04 11.08 73.27 123.13 0.41 0.80 3.73 59.96 0.24 0.50 1.97 0.65 9.84 0.46 31.53

1.53 510.26 2.94 15.64 736.45 0.09 18.08 1.71 0.35 1.88 0.55 0.05 1.65 1.17 1.93 1.69 0.95 3.12 24.88 52.27 0.05 0.11 0.76 30.85 0.03 0.02 0.39 0.07 0.95 0.06 4.08

Statistically significant.

rising serum CD4 counts and decreasing aqueous CMV viral load through the course of anti-CMV and HAART therapy suggests presence of a robust antiviral immune response. This study found that mediators involved in innate non-specific immune response and Th1-driven anti-viral response show a decreasing trend longitudinally through the course of treatment. The mediators involved in innate non-specific immune response include G-CSF, MCP-1, IL-8, IL-12p70 and IFN-g. The mediators in Th1-driven anti-viral response include IFN-g, IP-10, fractalkine and IL-10. Previous studies have reported similar results with regards to IL-8. One study reported that IL-8 was raised in human Retinal Pigment Epithelial cells (RPE) infected with CMV [18]. Another study reported raised levels of IL-8 and IL-10 in the aqueous of eyes with CMV retinitis, and that both IL-8 and IL-6 could be useful markers of treatment response [19]. Furthermore, studies of CMV in other cells also support our finding of elevation of MCP-1 in CMV infection. MCP-1 was found to be elevated in human fibroblasts in early stages of CMV infection [20]. There is also evidence to suggest a mechanism for CMV disruption of these cytokines; that it occurs via its own proteins such as pp71 and potentially contributes to viral pathogenesis by facilitating viral dissemination [20,21]. MCP-1 was also found to be elevated in a case-control observational murine study of transplanted infected infra-renal aorta grafts that were sampled at 2 and 8 weeks after transplantation for evaluation. They reported a 7fold increase in MCP-1 positive cells at 2 weeks, and a subsequent further 3.5-fold increase at 8 weeks [22]. However, contrary to findings in this study, a reciprocal relationship has been previously reported between levels of MCP-1 in CSF and the clinical status of viral meningoencephalitis [23]. This study conversely describes decreasing MCP-1 levels through treatment that correlated with decreasing local CMV viral load.

Other non-specific immune mediators such as RANTES, PDGFAA and Flt-3L also showed decreasing levels in association with decreasing CMV aqueous levels longitudinally in this study. Only aqueous PDGF-AA levels showed a negative correlation with systemic CD4 counts. Better demonstrable correlation of intraocular immune factor levels with CMV DNA load than with systemic CD4 counts suggests local CMV levels may be an important factor to consider in optimization of treatment regime for patients with CMVR. In one of our subjects with persistently depressed systemic CD4 levels –identified as the outlier – there was no significant change in immune factor levels through the course of treatment despite clinically quiescent disease. This could suggest subclinical inflammatory response that could result in a flare-up upon premature discontinuation of treatment, and a possibly higher risk of CMVR recurrence. The main limitation of this study is the small sample size with 17 patients, of whom 9 subjects were analyzed serially up to 12-weeks. Due to the small sample size and the relatively larger proportion of Chinese subjects and males, we were unable to correct for any possible confounding effect as a result of age, gender or race. The potential effect of this limitation on our results is further expounded by Mansfield et al. who studied variations of certain cytokines in normal ageing, and reported significant increase in MCP-1 and RANTES with age, and higher levels of PDGF-AA in women [24]. In addition, only 10 of the 17 subjects carried through with analysis of their aqueous humor through the course of their treatment and only 6 of the subjects had enough aqueous samples for longitudinal CMV DNA analysis. As such, the conclusions drawn from the study may not be definitive. Furthermore, a common dilution factor was required due to the nature of a multiplex assay, causing certain analytes to be outside of detection limits.

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Flt−3L

Fractalkine

100

G−CSF

100

10 10

10 1

Analyte concentration (pg/ml)

IFN−g

IL−8

IP−10 10000

100

10

100 1 1

MCP−1

PDGF−AA

RANTES

10000 10 10 1000 1

1

100

1

100

1

100

Virus load (copies/ul) Fig. 3. Scatter plot of CMV DNA (virus) load with analytes concentration in aqueous humor (pg/mL).

However, this is the first study to date that longitudinally tracks intraocular immune factor levels in CMVR through the course of treatment. These findings justify larger scale replication studies to examine the levels of these identified immune factors with specific testing in order to elucidate a ‘‘stop” point for treatment in patients with CMVR. This would facilitate individualization of treatment in CMVR, particularly in patients who develop CMVR during immunosuppression from etiologies other than HIV, such as post-organ transplantation. CD4 counts would not be relevant, and CMV viral loads are currently the only available measure of treatment response for these patients. In conclusion, this study identified immune factors indicative of recovery during treatment of CMVR. The end point of treatment in CMVR can be difficult to establish, and this study presents a potential novel solution and interesting new area of research. Conflicts of interest None to be declared.

Funding support National Medical Research Council (NMRC), New Investigator Grant (NIG), Singapore for Cytomegalovirus Retinitis Intravitreal Ganciclovir Singapore Study (CRIGSS), Grant Amount: SGD 1,99,900. References [1] X. Dagenais-Lussier, A. Mouna, J.P. Routy, et al., Current topics in HIV-1 pathogenesis: the emergence of deregulated immuno-metabolism in HIVinfected subjects, Cytokine Growth Factor Rev. (2015), http://dx.doi.org/ 10.1016/j.cytogfr.2015.09.001. pii:S1359-6101(15)30004-6 (Epub ahead of print). [2] M.W. Stewart, Optimal management of cytomegalovirus retinitis in patients with AIDS, Clin Ophthalmol. 4 (2010) 285–299. [3] D.A. Jabs, Ocular manifestations of HIV infection, Trans. Am. Ophthalmol. Soc. 63 (1995) 623–683. [4] B.D. Kupperman, J.G. Petty, D.D. Richman, et al., Correlation between CD4 counts and prevalence of cytomegalovirus retinitis and human immunodeficiency virus-related noninfectious retinal vasculopathy in patients with acquired immunodeficiency syndrome, Am. J. Ophthalmol. 115 (1993) 575–582.

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